Genetics and Molecular Biology, 31, 1, 166-171 (2008) Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br Short Communication Genetic structure in two northern muriqui populations (Brachyteles hypoxanthus, Primates, Atelidae) as inferred from fecal DNA Valéria Fagundes1, Marcela F. Paes1, Paulo B. Chaves1, Sérgio L. Mendes1, Carla de B. Possamai2,3, Jean P. Boubli4* and Karen B. Strier5 1Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, ES, Brazil. 2RPPN Feliciano Miguel Abdala, Caratinga, MG, Brazil. 3Pontifícia Universidade Católica de Minas Gerais, Belo Horizonte, MG, Brazil. 4Conservation and Research for Endangered Species, Zoological Society of San Diego, San Diego, CA, USA. 5Department of Anthropology, University of Wisconsin-Madison, Wisconsin, USA. Abstract We assessed the genetic diversity of two northern muriqui (Brachyteles hypoxanthus Primata, Atelidae) populations, the Feliciano Miguel Abdala population (FMA, n = 108) in the Brazilian state of Minas Gerais (19°44’ S, 41°49’ W) and the Santa Maria de Jetibá population (SMJ, n = 18) in the Brazilian state of Espírito Santo (20°01’ S, 40°44’ W). Fecal DNA was isolated and PCR-RFLP analysis used to analyze 2160 bp of mitochondrial DNA, made up of an 820 bp segment of the gene cytochrome c oxidase subunit 2 (cox2, EC 1.9.3.1), an 880 bp segment of the gene cytochrome b(cytb, EC 1.10.2.2) and 460 bp of the hypervariable segment of the mtDNA control region (HVRI). The cox2 and cytb sequences were monomorphic within and between populations whereas the HVRI revealed three different pop- ulation exclusive haplotypes, one unique to the SMJ population and two, present at similar frequencies, in the FMA population. Overall haplotype diversity (h = 0.609) and nucleotide diversity (π = 0.181) were high but reduced within populations. The populations were genetically structured with a high fixation index (FST = 0.725), possibly due to his- torical subdivision. These findings have conservation implications because they seem to indicate that the popula- tions are distinct management units. Key words: Brachyteles, conservation genetics, fecal DNA, mtDNA, PCR-RFLP. Received: April 27, 2007; Accepted: September 25, 2007. The muriqui or woolly spider monkey (Brachyteles The IUCN Red List cites the northern muriqui as a Spix 1823: Primates, Atelidae), endemic to Brazil, is the critically endangered species because only about 900 indi- largest Neotropical primate and was once widespread in the viduals are known dispersed in 12 populations, five of southeastern Atlantic Forest of Brazil. Aguirre (1971) esti- which contain less than 20 members. Some of the popula- mated a total population of 2,791-3,226 muriquis, contrast- tions are restricted to small, unprotected and isolated forest ing with a population of about 400,000 he reckoned would fragments extending from the south of the state of Bahia have existed in 1500. In the last decade this genus has been throughout Minas Gerais and Espírito Santo states as well split into two species (Groves, 2005), the northern muriqui as along the Mantiqueira Mountains (Serra da Mantiqueira) (Brachyteles hypoxanthus Kuhl 1820) and the southern on the borders of the states of Minas Gerais, Rio de Janeiro muriqui (Brachyteles arachnoides É. Geoffroy 1806). Cur- and São Paulo (Rylands et al., 2003b; Mendes et al., 2005). rent population estimates for the northern muriqui have The southern muriqui is distributed along the Serra do Mar indicated at least 864 individuals in the wild and data avail- from the south of Paraná state to Rio de Janeiro state, is able for the southern muriqui suggest a minimum popula- listed as endangered in the IUCN Red List and no more than tion of about 1,300 (Melo and Dias, 2005). 1300 are known to occur in relatively large and well pro- tected areas (Melo and Dias, 2005). Send correspondence to Valéria Fagundes. Laboratório de Gené- The small size and fragmented distribution of north- tica Animal, Departamento de Ciências Biológicas, Universidade ern muriqui populations compromises the ecological via- Federal do Espírito Santo, Av. Marechal Campos 1468, 29043-900 Maruípe, Vitória, ES, Brazil. E-mail: [email protected]. bility of this species (Brito and Grelle, 2006). Their *Present address: Department of Anthropology, The University of advanced age of 9 years at first reproduction and long Auckland, Auckland, New Zealand. inter-birth interval of three years, make the small persistent Fagundes et al. 167 populations more vulnerable to unfavorable demographic conditions than primates with faster life histories or larger populations (Strier et al., 2006). Habitat reduction and hunting have probably forced surviving northern muriqui populations into bottlenecks and reduced or eliminated op- portunities for gene flow through populations, resulting in changes in the frequency of alleles, loss of alleles, or both (Young and Clarke, 2000) similar to those documented in the golden lion tamarin (Grativol et al., 2001). Population viability analysis (PVA) has suggested that only muriqui populations of at least 700 monkeys would be genetically viable (Strier, 1993/1994; Brito and Grelle, 2006) but no empirical genetic data was included in these analyses. Although a preliminary allozyme analysis comparing one population of each species reported a high fixation index (Pope, 1998), knowledge of genetic structure of more populations is crucial for muriqui conservation planning (Fagundes, 2005). Standardization of a non-invasive method for DNA ex- traction from muriqui feces is an important tool for assessing the conservation status and behavioral ecology of these mon- keys (Chaves et al., 2006). Furthermore, data on the levels of genetic variability and differentiation in muriqui populations as well as pedigree reconstruction and information regarding Figure 1 - Map depicting the easternmost portion of Brazil. Sampling the relatedness between individuals and the extent of in- sites of the Brachyteles hypoxanthus populations are highlighted. The breeding can contribute to the planning of effective conser- FMA population in Minas Gerais (MG) state is roughly 150 km from the vation strategies for these species. municipality of Santa Maria de Jetibá (SMJ) in Espírito Santo (ES) state. We used the Polymerase Chain Reaction and Restric- tion Fragment Length Polymorphism analysis (PCR- man presence. The other population, designated the SMJ RFLP) to evaluate the genetic diversity of two northern population, is in privately owned forest fragments in the muriqui populations and characterize the distribution of ge- municipality of Santa Maria de Jetibá (SMJ) in the state of netic variability within and between populations. Fecal Espírito Santo (20°0’ S, 40°44’ W). The forest patches con- samples from 126 free-ranging northern muriquis taining the muriqui range from 60 ha to 350 ha and are (Brachyteles hypoxanthus Kuhl 1820) were collected im- highly fragmented due to agricultural activities and un- mediately after defecation and stored at 4 °C or room tem- evenly connected to each other by hilltop corridors. This perature (~24-28 °C) in 50 mL polypropylene vials contai- metapopulation has been estimated to be comprised of 115 ning a layer, about 1 mm to 4 mm deep, of desiccated silica muriqui (Mendes et al., 2005), although only about 16% beads to dehydrate the feces (Chaves et al. 2006). Each in- (n = 18) could be sampled because monitoring of this popu- dividual sampled was identified by its natural markings by lation only started in 2001. the experienced field researchers who collected the fecal We extracted DNA from the feces using the QIAamp samples within the ambit of a collecting license (number DNA Stool Minikit (Qiagen) and assessed the quality and 363/2001) issued by the Brazilian National Environmental amount using 1% (w/v) agarose gels stained with Agency (Instituto Brasileiro do Meio Ambiente e dos Re- 0.5 μg/mL ethidium bromide. Appropriate primers were cursos Naturais Renováveis – IBAMA). Two populations, used for the PCR amplification of a total of 2160 base pairs separated by 150 km, were studied (Figure 1). One popula- (bp) from the following three mitochondrial DNA tion is in a private conservation unit (Reserva Particular do (mtDNA) segments: 820 bp of the gene cytochrome c Patrimônio Natural Feliciano Miguel Abdala, RPPN- oxidase subunit 2 (cox2, EC 1.9.3.1), amplified using the FMA) of 957 hectares (ha) located in the state of Minas L6955/H7766 primer pair (Ashley and Vaughn, 1995); Gerais (19°44’ S, 41°49’ W) and is designated the FMA 880 bp of the gene cytochrome b (cytb, EC 1.10.2.2), ampli- population. This population is made up of four groups fied using the MVZ05/MVZ16 primer pair (Smith and (three mixed-sex and one only-male group) totaling 226 Patton, 1993); and the 460 bp hypervariable segment of the northern muriquis (Strier et al., 2006) of which nearly 50% mtDNA control region (HVRI), for which we designed a (n = 108) were sampled between 2001 and 2002, once this novel specific primer pair consisting of a 5’-CTACTCCCT population has been monitored over the last 25 years by K. GAATAACCAAC-3’ forward primer (Mono1) and a 5’- B. Strier and her coworkers and animals are used to the hu- AGCGAGAAGAGCGGCAAATG-3’ reverse primer 168 Genetic structure of northern muriqui (Mono2), which were based on the Brachyteles sequence For the restriction fragment data analysis, one allele (GenBank AF213966) with the 3’ annealing positions was represented by one fragment obtained with an enzyme, (L15463 for Mono1 and H15890 for Mono2) from the and the set of all alleles of a specific digestion (i.e., the Cebus albifrons mtDNA sequence (GenBank AJ309866). cleavage pattern) received a capital letter (Table 1). The The specificity of the HVRI primers was shown by their in- composite haplotype panel for each monkey included the ability to amplify human DNA, probably due to the 19 mis- cleavage pattern of all the enzymes (Table 2, see Bates, matches (12 forward and 7 reverse) between the primers, 2002 for details).